Process for the production of acetone.



g ITED STATE FATE @FQ.

, JOHN H. NORTH ROP, OF NEW YORK, N. Y., ASSIGNOR TO THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH, 01? NEW YORK, N. 'Y., A CORPORATION OF NEW YORK.

PROCESS FOR THE PRODUCTION OF ACETONE.

No Drawing.

To all whom it may concern:

Be it known that I, J OHN H. Non'rnsor, a

citizen of the United States, and a resident lowing is a specification.

The process has for its object the production of acetone, and incidentally ethyl alco- 1101, from products containing starches,

sugar and. similar carbohydrates, such as corn, molasses, potatoes, horse-chestnuts,

etc. In carrying out my process the starchy or sugary material is subjected to fermentat1on by the act1on of a certain bacillus acetoethylicum.

The bacillus-ethylicum was obtained from a sample of old potatoes. According to thedescription chart of the'Society of American Bacteriologist-s, said bacillus is described as follows: ourae.-Old potatoes. 1 Date of isolation-July, 1917.

N (mgr-Bacillus .aceto-ethylicum.

I. Morphology. r 1. Vegetative cells, motile:

(a) Media used+-a ar slant, (if 10% corn media l'part corn in 10 parts water), temp. 40 O age 24 hours.

-Forin'short rods, no chains in agar slant. vOccasional short chains in '10% cornmedia.

Size'-4-6p.-.X0.2-0.3p.. Ends rounded.

Stain -evenly with 'Loefliers meth-j Patented Feb. 4, 1912.

Application filed m s, 1918. Serial No. 243,811.

2. Sporangia:

" ylene blue or "gentianviolet.

- Gram stain negat ve. v (b) Media used10% corn 1- media,

' temp. 40 0., age6l0 days.

Form hort rods..- occasional short chains. A q Size-+6 .X0.2-0.3 Ends-rounded. j Stain-unevenly methylene blue or gentian violet, with deep stain spot at end or in center. Gram stain negative.-

. Glucose broth. After 24 hours,'40 C.

. Litmus milk, 24 hours, 40 vC. 4 -Bottom of tube reduced to white, no

- 72 hours, 40 C. Same but coagulated;

Media used, glucose agar slant.

Temperature40 C., after 2 to 3 days spores formed.

Form'elliptical. Spores form at end of rods.

Limits of size 1p..

Size of majority-.6.8p..

Spores stain easily with Loefllers methylene blue.

1. Cultural features.

2% glucose agar slant media, age 24 hours, temp. 40 C. Growth, moderate. Form of growth, spreading. Elevation of growth, eifus Luster, dull. I .Optical character, translucent. Odor, absent. Media, clear.

Condensation water, opaque.

Potato, 24 hours, 40? 0.. Nowisi-ble growth on surface.

,Gasbubbles all over media, crumbles easily, no odor. Aftef2-3 days, 40 C. Media sinks to grayish white paste. so

(Media cloud no odor.)

- Slight gas bubb es.

After 2-3 days, slimy mass in bottom of test tube.

gas,odor, acid, nor clot. 36 hours, 40? C. Milk red on top, rest White. i i

clot does not digest subsequently.

Agar colonies. 2-per cent. glucose-agar, 24 hours, 40 C.

Growth, slowly spreadin I Round, sometimes irregular.

- Surface, smooth.

Elevation, efiuse.

Edge entire or undulate,

Internal structure, coarsely granular.

H. Cultural featu-res.0ontd.

Sodium chlorid in bouillon.

Inhibiting concentration is 4-5 per cent.

sodium chlorid.

Nitrogen source. With sugar as carbohydrate may be obtained from peptone, proteins or ammonium salts.

With starch. Same, but cannot use ammonium salts.

Best media for long continued growth.

2 per cent. corn media with GaCo [1]. Physical and biochemical features.

1 V. Pathogem'oitg). 1. Non-pathogenic to mice.

In carrying out my process, a mash of the substance to be fermented is prepared, con- Sorensens taming preferably 2 to 10 parts of-carbohydrate material, 100 parts water, and about 11; t0 1 part of some suitable nitrogenous 'material, such as protein, if sufficient is not already contained in the carbohydrate material used. This mash is then sterilized, preferably by heat, and is cooled to a temperature of from 35 to 45 (1., and the reaction brought ,to a hydrogen ion concentration of 6.8 to 7.4: (Sorensen H values) by means of some suitable soluble or insoluble buffer substance, such as calcium carbonate or sodium phosphate, which willprevent the mash from becoming acid. The mash is then put into a closed sterile vessel, preferably filled with some inert material, such as marble chips or brush, and inoculated with a culture of the bacillus aceto-ethylicum above described, and allowed to ferment for from 4: to

8 .days. At the end of that time some 8% to 13% of the dry weight of the carbohydrate has been converted into acetone; and

about 18% to 26% of the dry weight of the carbohydrate has been converted into alco- 110 1, principally ethyl alcohol. The fermented material treated is then removed from the vessel and the alcohol and acetone are recovered by any well known method, such as distillation. A fresh supply of mash prepared in the same way may be put into the vessel and is caused to ferment by the bacilli adhering to the walls of the Vessel and the inert material after the withdrawal of the first batch of fermented material. The second and succeeding fermentations are somewhat more rapid than the'first one, the second and succeeding operations being completed in from 40 to hours.

The amount of alcohol and acetone which is produced by the above process varies with the kinds of carbohydrate material employed. The following table, which was prepared from laboratory experiments con-- ducted by using the materials named in a 1% solution with the addition of a suitable amount of calcium carbonate as a buffer substance and of peptone as a nitrogen source, illustrates the relative yield of acetone and alcohol obtained from the various substances named. Except as otherwise specified, the

percentages refer to the dry weight of the substance used.

* Vol. per cent.

** Per cent. 01 fresh weight Ferments also under following conditions, with same percenta e yield of acetone and alcohol:

M'e ia.Water, 1 liter; Potassium phosphate, 1 gram; ammonium phosphate, 1 gram; sodium chlond, trace; calcium carbonate, 2-5 grams; galatose or levulose, 10 grams. Fermented at 40 0., in Grubers anaerobic tubes-fermentation complete in 6-7 days.

Dextrose, levulose, galactose, cane sugar (sucrose), maltose,

Table of sugars fermented.

Per cent. Per cent. Substance acetone. alcohol.

- Galactose 4- 5 22-24 Maltose 6- 7 23-24 Mannose 6- 7 22-23 Raffinose... 8-10 22-23 d-Arabinose. 6- 7 12-16 Car-lactate. Starch 8-10 20-24 Beet molasses *8-10 ut-24 Potatoes, white or swee 2- 4 4- 9 Dextrin 7 14-16 Dextrose 9-10 22-23 Levulose 8-10 24-25 Xylose 4- 5 18-20 Glycerin 40-43 Sucrose 8- 9 24-26 cm 10-13 20-26 Corn cobs 1- 5 8-10 Horse chestnuts. 7- 8 14-17 lactose, starch, d-arabinose also ferment with the above solution in a flask of such size that the liquid forms a shallow layer on the bottom.

The following are examples of specific operations: I. A mash was prepared, consisting of 5 kilograms of corn, 5/10 kilogram of autolyzed yeast, 5/10 kilogram calcium carbonate and 100 kilograms of water. The mash was sterilized by heating under 15 lbs. steam pressure (about C.) for about four hours. The mash was then put into a sterilized tank filled with chipped marble and inoculated with a pure culture of the abovedescribed bacillus. After four days, analysis of a sample of the material showed the presence of acetone to the extent of 9% of the dry weight of the corn. The fermented material was then drawn off from the tank and a fresh mash, prepared like the first,

\ with the bacilli remaining from the pre- 'vious run. The fermentation was completed in about 40 hours and the yield of acetone was approximately the same 'as in the first run after four days. This operation was then repeated several times. In each case "the acetone and alcohol were recovered: by

distillation. The yield of the acetone in the several runs varied between 8% and 10% of the dry weight of the corn and the yield of ethyl alcohol varied between 12% and 20% of the dry weight of the corn.

-11. A mixture was prepared, consisting of molasses diluted with water so as to contain 8% of sugar. The mixture was sterilized by heating under 15 lbs. steam pressure (about 115 C.) for about four hours. The mixture was then put into a sterilized tank filled with chipped marble and inoculated'with a pure culture 'of the above-described bacillus. ter four days, analysis of a sample of the material showed the presence of acetone to V the extent of 9% of sugar content. The fermented material was then drawn off from the tank and a fresh mixture, prepared like the first, was put into it and automatically-inoculated with the bacilli remaining from the previous run. The fermentation was completed in about 40 hours and the yield of acetone was approximately the same as in the first run after four days.

' This operation was then repeated several times. In each case the acetone and alcohol were recovered by distillation. The yield of 'the acetone in the several runs varied between 8% and 10% of sugar content and the yield of ethyl alcohol varied between 12% and 20% of the sugar content. I In treating molasses the addition of nitrogenous unnecessary,

growth of the bacilli. This is a so truein the treatment of other carbohydrate mate'- rials which contain nitrogenous matter in .sutficient quantities.

, closed vessel. In' carrying out of coursebe taken Again, treating molasses,

it is not necessary to add bufi'er material to the mixture prior to putting it into the the process, care should It is obvious that various changes may be made in the details of my processes without r'naterial with the herein-described bacillus aceto-ethylicum.

material 'tothe mixture is usually because the molasses contains sufficient nitrogenous material to ermit the.

if marble chips are used as inert masses in the closed vessel,

to avoid contaminating thejmash or mixture-with inoculations of. other bacilli or organisms after sterilization.

nitrogenous material and a bufier substance,

with the herein-described bacillus acetoethylicum.

4:. The process of producing acetone,

which comprlses preparing a mixture of carbohydrate material and water, containing also some nitrogenous material and some buffer substance, sterilizing the mixture, inoculatin scribed mitting the mixture to ferment by the action of said bacillus.

5. The process 'of producing acetone, which comprises preparing amixture of carbohydrate material and water, containing also some nitrogenous material and some buffer substance, sterilizing the mixture, put tingthe mixture in a closed vessel, inoculating the mixture. with the herein-described bacillus aeeto-ethylicum, and permitting the mixture-to ferment by the action of said bacillus.

6. The process of producing acetone, which comprises preparing a mixture of carbohydrate material and water, containing also some nitrogenous material and some buffer substance, sterilizing the m1xture,put-

inert materialfmoculating the mixture with the herein-described bacillus a'ceto-ethylicum, and permitting the mixture to ferment by the action "of said bacillus.

7. The process of producing acetone, which comprises fermenting molasses with the herein-described bacillus aceto-ethylicum.

8. The process of producing acetone, which comprises fermenting molasses in the presence of a buffer substance with the here- 'in-described bacillus aceto-ethylicum.

which comprises preparing a mixture of molasses and water, adding some buffer substance, sterilizing the mixture, inoculating the mixture with the herein-described bacillus aceto-ethylicum, and permitting the mix- .ture' to ferment by the action of said bacillus. s

10. The process of producing acetone, which comprises preparing a mixture of molasses and water, adding some buifer substance, sterilizing the mixture putting the mixture in a closed vessel, inoculating the mixture with the herein-described bacillus acto-ethyhcum, and permitting the mixture to ferment by the action of said bacillus.

1 1;The process of producing acetone,

the mixture with the herein-deacillus aceto-ethylicum, and per-- 9. The process of producing acetone,

f/ting the mixture in a closed vessel contain- ..1n masses of bohydrate material and water,

which comprisespreparing a mixture of molasses and water, adding some bufi'er substance, SteriliZing the mlxture, putting the mixture in a closed vessel containing masses of inert material, inoculating the mixture with the herein-described bacillus acetoethylicum, and permitting the mixture to ferment by the action of the said bacillus.

12. The process of producing acetone, which comprises preparing a mixture of carbohydrate mater1al and Water, containing also some nitrogenous material and some bufl'er substance, sterilizing the mixture, putting the mixture in a closed vessel, inoculating the mixture with the herein-described bacillus aceto-ethylicum, permitting the mixture to ferment by the action of said bacillus, withdrawing the fermented material from the vessel, and putting into the vessel a similarly prepared fresh mixture and permitting it to ferment.

13. The process of producing acetone, which comprises preparing a mixtureof carcontaining also some nitrogenous material and some bufier substance, sterilizin the mixture, putting the mixture in a closed masses of inert material, inoculating the mixture with the herein-described bacillus aceto-ethylicum, permitting the mixture to vessel containing ferment by the action of said bacillus, withdrawing the fermented material from the vessel, and putting into the vessel a similarly prepared fresh mixture and permitting it to ferment.

14. The process of producing acetone, which comprises preparing a mixture of molasses and water, adding some buffer substance, sterilizing the mixture, inoculating the mixture with the herein-described bacillus aceto-ethylicum, permitting the mixture to ferment by the action of said bacillus, withdrawing the fermented material from the vessel, and putting into the vessel a similarly prepared fresh mixture and permitting it toferment.

15. The process of producing acetone, which comprises preparing a mixture of molasses and water, adding some buffer substance, sterilizing the mlxture, putting the mixture in a closed vessel, inoculating the mixture with the herein-described bacillus aceto-ethylicum, permitting the mixture to ferment by the action of said bacillus, withdrawing the fermented material from the vessel, and putting into the vessel a similarly prepared fresh mixture and permitting it to feent.

JOHN H. N ORTHROP. 

